Quality control and filtering results from cellranger

Sample info and environment setup

PRJNA732205

setwd("/media/jacopo/Elements/re_align/MM/PRJNA732205/SAMN19314095/SRR14629349/")
# Load the libraries (from Sarah script + biomart)
library(tidyverse) # packages for data wrangling, visualization etc
library(Seurat) # scRNA-Seq analysis package
library(clustree) # plot of clustering tree 
library(ggsignif) # Enrich your 'ggplots' with group-wise comparisons
library(clusterProfiler) #The package implements methods to analyze and visualize functional profiles of gene and gene clusters.
library(org.Hs.eg.db) # Human annotation package neede for clusterProfiler
library(ggrepel) # extra geoms for ggplo2
library(patchwork) #multiplots
library(reticulate)

Load and process cellranger data

Load and do the QC for the cellranger data

#list.files(".")
dat <- Read10X(data.dir ="./out/counts_filtered/")
dat <- CreateSeuratObject(dat) # Create the seurat object from the 10x data
kb.initial <- dat@assays[["RNA"]]@counts@Dim[[2]]
cat("Initial number of cells:", kb.initial, 
    "\nNumber of genes:",  dat@assays[["RNA"]]@counts@Dim[[1]])
## Initial number of cells: 9769 
## Number of genes: 36601

Quality Control

Empty cells were already filtered, check for % mt RNA and death markers:

# first calculate the mitochondrial percentage for each cell
dat$percent_mt <- PercentageFeatureSet(dat, pattern="^MT.")
# make violin plots
mt_rna = 20
max_counts = 12000



# Check some feature-feature relationships
# % mt RNA vs n Counts, n Features vs n Counts
# Check some feature-feature relationships
# % mt RNA vs n Counts, n Features vs n Counts
VlnPlot(dat, features = c("nCount_RNA", "nFeature_RNA", "percent_mt"))  + geom_hline(yintercept=mt_rna, linetype = "dotted")

plot1 <- FeatureScatter(dat, feature1 = "nCount_RNA", feature2 = "percent_mt")
plot1 <- plot1 + geom_hline(yintercept=mt_rna, linetype = "dotted")
plot2 <- FeatureScatter(dat, feature1 = "nCount_RNA", feature2 = "nFeature_RNA")
plot2 <- plot2 + geom_vline(xintercept = max_counts, linetype = "dotted")
plot1 

plot2

##  cells retained by mt RNA content ( 20 %): 7308 
##  percentage of retained cells: 74.81 %
## cells retained by counts ( 12000 ): 7288 
##  percentage of retained cells: 74.6 %

Check the distribution of the cells with low counts and control death markers:

min_counts = 200


hist(dat@meta.data$nCount_RNA, breaks = 100, xlab = "Counts")

hist(dat@meta.data$nCount_RNA, breaks = 100, xlab = "Counts", xlim = c(0,5000))

hist(dat@meta.data$nCount_RNA, breaks = 1000, xlab = "Counts", xlim = c(0,1000))
abline(v=min_counts, col="red", lty = 3)

The evident peak of cells with < 200 counts could contain dying cells.

# Subset the dataset to focus only on those cells with low counts
dat.lowcount <- subset(dat, subset = nCount_RNA < min_counts)

# Get the mean of the counts for each gene and sort them decreasing
meanCounts <- rowMeans(GetAssayData(object = dat.lowcount, slot = 'counts'))
meanCounts <- sort(meanCounts, decreasing = T)

# A boxplot can help to observe the distribution of the means
#boxplot(meanCounts)

# Print the most highly expressed genes
head(meanCounts, 30)
##      IGKC     IGHG1  IGHV4-61    JCHAIN    MT-CO2     IGHG3     RPLP1    MALAT1 
## 9.4219653 6.8815029 5.2803468 4.8294798 3.4653179 2.5433526 1.8497110 1.7745665 
##       B2M     IGHG4    MT-ND3 MTRNR2L12     RPL34     RPS28   MT-ND4L     RPL39 
## 1.3786127 1.3699422 1.1213873 1.0809249 0.9884393 0.9161850 0.9161850 0.9132948 
##   MT-ATP6     RPL32     RPS12    EEF1A1      RPS8     RPL41    MT-CYB      SSR4 
## 0.8381503 0.7167630 0.7109827 0.6936416 0.6618497 0.6416185 0.6416185 0.6213873 
##    RPL18A    MT-CO3    MT-ND5     RPL10     RPS19    MT-CO1 
## 0.6156069 0.5953757 0.5491329 0.5317919 0.5289017 0.5260116
## cells retained by counts ( 200 ): 6942 
##  percentage of retained cells: 71.06 %

dir.create("result")
saveRDS(dat, file = "./result/SAMN19314095_clean_QC.Rds")

Feature selection

#Normalize
dat <- NormalizeData(dat)
# Find the first 4000 variabe features
dat <- FindVariableFeatures(dat, selection.method = "vst", nfeatures = 4000)

Data scaling

Set mean expression to 0 and variance across 1 to avoid highly expressed genes drive the forwarding analyses. Since negative expression is meaningless, scaled data are useful only for UMAP and clustering

# scale data, the scaled data are saved in:
# dat[["RNA"]]@scale.data

all.genes <- rownames(dat)

dat <- ScaleData(dat, vars.to.regress = c("percent_mt","nCount_RNA"))

Dimensionality reduction

dat <- RunPCA(dat, features = VariableFeatures(object = dat), verbose = F, seed.use = 1)
print(dat[["pca"]], dims = 1:5, nfeatures = 5)
## PC_ 1 
## Positive:  IGKC, IGHG1, IGHV4-61, JCHAIN, IGHG3 
## Negative:  IGLL1, STMN1, TYMS, PCLAF, VPREB1 
## PC_ 2 
## Positive:  MS4A1, CD52, HLA-DQB1, HLA-DPA1, HLA-DRB1 
## Negative:  IGHG1, IGHG3, TYMS, IGKC, IGLL1 
## PC_ 3 
## Positive:  FOS, GADD45B, JUNB, HIST1H2AC, HIST1H2AE 
## Negative:  B2M, IGHA2, CD27, IGHV3-23, H2AFZ 
## PC_ 4 
## Positive:  CD27, IGHA2, IGHG2, IGHV3-23, IGHA1 
## Negative:  IGHV4-61, JCHAIN, IGHV4-34, IGKV1-5, PTMA 
## PC_ 5 
## Positive:  AFF3, TCL1A, TOP2A, CFAP73, RUBCNL 
## Negative:  HCST, S100A4, AIF1, TYROBP, LST1

UMAP

UMAP is a graph-based method of clustering. The first step in this process is to construct a KNN graph based on the euclidean distance in PCA space:

dat <- FindNeighbors(dat, dims = 1:20)

The graph now can be used as input for the function runUMAP()

dat <- RunUMAP(dat, dims = 1:20, seed.use = 1)
DimPlot(dat, reduction = 'umap', seed = 1)

Final plots:

## QC metrics

## markers